This invention relates to the identification of antimicrobial agents and of microbial targets of such agents, and in particular to the isolation of bacteriophage DNA sequences, and their translated protein products, showing anti-microbial activity. The DNA sequences can be expressed in expression vectors. These expression constructs and the proteins produced therefrom can be used for a variety of purposes including therapeutic methods and identification of microbial targets.
The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the present invention.
The frequency and spectrum of antibiotic-resistant infections have, in recent years, increased in both the hospital and community. Certain infections have become essentially untreatable and are growing to epidemic proportions in the developing world as well as in institutional settings in the developed world. The staggering spread of antibiotic resistance in pathogenic bacteria has been attributed to microbial genetic characteristics, widespread use of antibiotic drugs and changes in society that enhance the transmission of drug-resistant organisms (for a review, see Cohen, 1992). This spread of drug resistant microbes is leading to ever-increasing morbidity, mortality and health-care costs.
There are over 160 antibiotics currently available for treatment of microbial infections, all based on a few basic chemical structures and targeting a small number of metabolic pathways: bacterial cell wall synthesis, protein synthesis, and DNA replication. Despite all these antibiotics, a person could succumb to an infection as a result of a resistant bacterial infection. Resistance now reaches all classes of antibiotics currently in use, including: xcex2-lactams, fluoroquinolones, aminoglycosides, macrolide peptides, chloramphenicol, tetracyclines, rifampicin, folate inhibitors, glycopeptides, and mupirocin. There is thus a need for new antibiotics, and this need will not subside given the ability bacteria have to overcome each new agent synthesized. It is also likely that targeting new pathways will play an important role in discovery of these new antibiotics. In fact, a number of crucial cellular pathways, such as secretion, cell division, and many metabolic functions, remain untargeted today.
Most major pharmaceutical companies have on-going drug discovery programs for novel anti-microbials. These are based on screens for small molecule inhibitors (e.g., natural products, bacterial culture media, libraries of small molecules, combinatorial chemistry) of crucial metabolic pathways of the micro-organism of interest. The screening process is largely for cytotoxic compounds and in most cases is not based on a known mechanism of action of the compounds. Classical drug screening programs are being exhausted and many of these pharmaceutical companies are looking towards rational drug design programs. Several small to mid-size biotechnology companies, as well as large pharmaceutical companies, have developed systematic high-throughput sequencing programs to decipher the genetic code of specific micro-organisms of interest. The goal is to identify, through sequencing, unique biochemical pathways or intermediates that are unique to the microorganism. Knowledge of the function of these bacterial genes, may form the rationale for a drug discovery program based on the mechanism of action of the identified enzymes/proteins. However, one of the most critical steps in this approach is the ascertainment that the identified proteins and biochemical pathways are 1) non-redundant and essential for bacterial survival, and 2) constitute suitable and accessible targets for drug discovery. These two issues are not easily addressed since to date, 18 prokaryotic genomes have been sequenced and 200 sequenced genomes are expected by the year 2000. For a majority of the sequenced genomes, less than 50% of the open reading frames (ORFs) have been linked to a known function. Even with the genome of Escherichia coli (E. coli), the most extensively studied bacterium, less than two-thirds of the annotated protein coding genes showed significant similarity to genes with ascribed functions (Rusterholtz and Pohlschroder, 1999). Thus considerable work must be undertaken to identify appropriate bacterial targets for drug screening.
The present invention is based on the identification of, and demonstration that, specific DNA sequences of a bacteriophage, when introduced into a host bacterium can kill, or inhibit growth, of the host. Thus, these DNA sequences are anti-microbial agents. Information based on these DNA sequences can be utilized to develop peptide mimetics that can also function as anti-microbials. The identification of the host bacterial proteins, targeted by the anti-microbial bacteriophage DNA sequences, can provide novel targets for drug design and compound screening.
In this regard, the terms xe2x80x9cinhibitxe2x80x9d, xe2x80x9cinhibitionxe2x80x9d, xe2x80x9cinhibitoryxe2x80x9d, and xe2x80x9cinhibitorxe2x80x9d all refer to a function of reducing a biological activity or function. Such reduction in activity or function can, for example, be in connection with a cellular component (e.g., an enzyme), or in connection with a cellular process (e.g., synthesis of a particular protein), or in connection with an overall process of a cell (e.g., cell growth). In reference to cell growth, the inhibitory effects may be bactericidal (killing of bacterial cells) or bacteriostatic (i.e., stopping or at least slowing bacterial cell growth). The latter slows or prevents cell growth such that fewer cells of the strain are produced relative to uninhibited cells over a given time period. From a molecular standpoint, such inhibition may equate with a reduction in the level of, or elimination of, the transcription and/or translation of a specific bacterial target(s), or reduction or elimination of activity of a particular target biomolecule.
In a first aspect the invention provides methods for identifying a target for antibacterial agents by identifying the bacterial target(s) of at least one inhibitory gene product, e.g., protein from ORFs 33, 41, 79 of bacteriophage 3A, ORF 1 of bacteriophage 77 and ORFs 48, 78, 100 of bacteriophage 96 or a homologous product. Such identification allows the development of antibacterial agents active on such targets. Preferred embodiments for identifying such targets involve the identification of binding of target and phage ORF products to one another. The target molecule may be a bacterial protein or other bacterial biomolecule, e.g., a nucleotprotein, a nucleic acid, a lipid or lipid-containing molecule, a nucleoside or nucleoside derivative, a polysaccharide or polysaccharide-containing molecule, or a peptidoglycan. The phage ORF products may be subportions of a larger ORF product that also binds the host target. Exemplary approaches are described below in the Detailed Description.
Additionally, the invention provides methods for identifying targets for antibacterial agents by identifying homologs of a Staphylococcus aureus target of a bacteriophage 3A ORF product, for example, ORFs 33, 41 or 79, bacteriophage 77 ORF product, such as for example, ORF 1 or bacteriophage 96 ORF products, such as for example, ORFs 48, 78, or 100 product. Such homologs may be utilized in the various aspects and embodiments described herein.
The term xe2x80x9cfragmentxe2x80x9d refers to a portion of a larger molecule or assembly. For proteins, the term xe2x80x9cfragmentxe2x80x9d refers to a molecule which includes at least 5 contiguous amino acids from the reference polypeptide or protein, preferably at least 6, 8, 10, 12, 15, 20, 30, 50 or more contiguous amino acids. In connection with oligo- or polynucleotides, the term xe2x80x9cfragmentxe2x80x9d refers to a molecule which includes at least 15 contiguous nucleotides from a reference polynucleotide, preferably at least 18, 21, 24, 30, 36, 45, 60, 90, 150, or more contiguous nucleotides. Also in preferred embodiments, the fragment has a length in a range with the minimum as described above and a maximum which is no more than 90% of the length (or contains that percent of the contiguous amino acids or nucleotides) of the larger molecule (e.g., of the specified ORF), in other embodiments, the upper limit is no more than 60, 70, or 80% of the length of the larger molecule.
Stating that an agent or compound is xe2x80x9cactive onxe2x80x9d a particular cellular target, such as the product of a particular gene, means that the target is an important part of a cellular pathway which includes that target and that the agent acts on that pathway. Thus, in some cases the agent may act on a component upstream or downstream of the stated target, including a regulator of that pathway or a component of that pathway. In general, an antibacterial agent is active on an essential cellular function, often on a product of an essential gene.
By xe2x80x9cessentialxe2x80x9d, in connection with a gene or gene product, is meant that the host cannot survive without, or is significantly growth compromised, in the absence or depletion of functional product. An xe2x80x9cessential genexe2x80x9d is thus one that encodes a product that is beneficial, or preferably necessary, for cellular growth in vitro in a medium appropriate for growth of a strain having a wild-type allele corresponding to the particular gene in question. Therefore, if an essential gene is inactivated or inhibited, that cell will grow significantly more slowly or even not at all. Preferably growth of a strain in which such a gene has been inactivated will be less than 20%, more preferably less than 10%, most preferably less than 5% of the growth rate of the wild-type, or not at all, in the growth medium. Preferably, in the absence of activity provided by a product of the gene, the cell will not grow at all or will be non-viable, at least under culture conditions similar to normal in vivo growth conditions. For example, absence of the biological activity of certain enzymes involved in bacterial cell wall synthesis can result in the lysis of cells under normal osmotic conditions, even though protoplasts can be maintained under controlled osmotic conditions. Preferably, but not necessarily, if such a gene is inhibited, e.g., with an antibacterial agent or a phage product, the growth rate of the inhibited bacteria will be less than 50%, more preferably less than 30%, still more preferably less than 20%, and most preferably less than 10% of the growth rate of the uninhibited bacteria. As recognized by those skilled in the art, the degree of growth inhibition will generally depend on the concentration of the inhibitory agent. In the context of the invention, essential genes are generally the preferred targets of antimicrobial agents. Essential genes can encode target molecules directly or can encode a product involved in the production, modification, or maintenance of a target molecule.
A xe2x80x9ctargetxe2x80x9d refers to a biomolecule that can be acted on by an exogenous agent, thereby modulating, preferably inhibiting, growth or viability of a cell. In most cases such a target will be a nucleic acid sequence or molecule, or a polypeptide or protein. However, other types of biomolecules can also be targets, e.g., membrane lipids and cell wall structural components.
The term xe2x80x9cbacteriumxe2x80x9d refers to a single bacterial strain, and includes a single cell, and a plurality or population of cells of that strain unless clearly indicated to the contrary. In reference to bacteria or bacteriophage, the term xe2x80x9cstrainxe2x80x9d refers to bacteria or phage having a particular genetic content. The genetic content includes genomic content as well as recombinant vectors. Thus, for example, two otherwise identical bacterial cells would represent different strains if each contained a vector, e.g., a plasmid, with different phage ORF inserts.
In the context of the phage nucleic acid sequences, e.g., gene sequences, of this invention, the terms xe2x80x9chomologxe2x80x9d and xe2x80x9chomologousxe2x80x9d denote nucleotide sequences from different bacteria or phage strains or species or from other types of organisms that have significantly related nucleotide sequences, and consequently significantly related encoded gene products, preferably having related function. Homologous gene sequences or coding sequences have at least 70% sequence identity (as defined by the maximal base match in a computer-generated alignment of two or more nucleic acid sequences) over at least one sequence window of 48 nucleotides (or at least 99, 150, 200, or even the entire ORF or other sequence of interest), more preferably at least 80 or 85%, still more preferably at least 90%, and most preferably at least 95%. The polypeptide products of homologous genes have at least 35% amino acid sequence identity over at least one sequence window of 18 amino acid residues (or 24, 30, 33, 50, 100, or an entire polypeptide), more preferably at least 40%, still more preferably at least 50% or 60%, and most preferably at least 70%, 80%, or 90%. Preferably, the homologous gene product is also a functional homolog, meaning that the homolog will functionally complement one or more biological activities of the product being compared. For nucleotide or amino acid sequence comparisons where a homology is defined by a % sequence identity, the percentage is determined using BLAST programs (with default parameters (Altschul et al., 1997, xe2x80x9cGapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acid Res. 25:3389-3402). Any of a variety of algorithms known in the art which provide comparable results can also be used, preferably using default parameters. Performance characteristics for three different algorithms in homology searching is described in Salamov et al., 1999, xe2x80x9cCombining sensitive database searches with multiple intermediates to detect distant homologues.xe2x80x9d Protein Eng. 12:95-100. Another exemplary program package is the GCG(trademark) package from the University of Wisconsin.
Homologs may also or in addition be characterized by the ability of two complementary nucleic acid strands to hybridize to each other under appropriately stringent conditions. Hybridizations are typically and preferably conducted with probe-length nucleic acid molecules, preferably 20-100 nucleotides in length. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower complementarity will not. For examples of hybridization conditions and parameters, see, e.g.,. Maniatis, T. et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor University Press, Cold Spring, N.Y.; Ausubel, F. M. et al. (1994) Current Protocols in Molecular Biology. John Wiley and Sons, Secaucus, N.J. Homologs and homologous gene sequences may thus be identified using any nucleic acid sequence of interest, including the phage ORFs and bacterial target genes of the present invention.
A typical hybridization, for example, utilizes, besides the labeled probe of interest, a salt solution such as 6xc3x97SSC (NaCl and Sodium Citrate base) to stabilize nucleic acid strand interaction, a mild detergent such as 0.5% SDS, together with other typical additives such as Denhardt""s solution and salmon sperm DNA. The solution is added to the immobilized sequence to be probed and incubated at suitable temperatures to preferably permit specific binding while minimizing non-specific binding. The temperature of the incubations and ensuing washes is critical to the success and clarity of the hybridization. Stringent conditions employ relatively higher temperatures, lower salt concentrations, and/or more detergent than do non-stringent conditions. Hybridization temperatures also depend on the length, complementarity level, and nature (i.e., xe2x80x9cGC contentxe2x80x9d) of the sequences to be tested. Typical stringent hybridizations and washes are conducted at temperatures of at least 40xc2x0 C., while lower stringency hybridizations and washes are typically conducted at 37xc2x0 C. down to room temperature (xcx9c25xc2x0 C.). One of ordinary skill in the art is aware that these conditions may vary according to the parameters indicated above, and that certain additives such as formamide and dextran sulphate may also be added to affect the conditions.
By xe2x80x9cstringent hybridization conditionsxe2x80x9d is meant hybridization conditions at least as stringent as the following: hybridization in 50% formamide, 5xc3x97SSC, 50 mM NaH2PO4, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5xc3x97Denhart""s solution at 42xc2x0 C. overnight; washing with 2xc3x97SSC, 0.1% SDS at 45xc2x0 C.; and washing with 0.2xc3x97SSC, 0.1% SDS at 45xc2x0 C. In another example, stringent hybridization conditions should not allow for hybridization of two nucleic acids which differ over a stretch of 20 contiguous nucleotides by more than two bases.
Homologous nucleotide sequences will distinguishably hybridize with a reference sequence with up to three mismatches in ten (i.e., at least 70% base match in two sequences of equal length). Preferably, the allowable mismatch level is up to two mismatches in 10, or up to one mismatch in ten, more preferably up to one mismatch in twenty. (Those ratios can, of course, be applied to larger sequences.)
Preferred embodiments involve identification of binding between ORF product and bacterial cellular component that include methods for distinguishing bound molecules, for example, affinity chromatography, immunoprecipitation, crosslinking, and/or genetic screen methods that permit protein:protein interactions to be monitored. One of skill in the art is familiar with these techniques and common materials utilized (see, e.g., Coligan, J. et al. (eds.) (1995) Current Protocols in Protein Science, John Wiley and Sons, Secaucus, N.J.).
Genetic screening for the identification of protein:protein interactions typically involves the co-introduction of both a chimeric bait nucleic acid sequence (here, the phage ORF to be tested) and a chimeric target nucleic acid sequence that, when co-expressed and having affinity for one another in a host cell, stimulate reporter gene expression to indicate the relationship. A xe2x80x9cpositivexe2x80x9d can thus suggest a potential inhibitory effect in bacteria. This is discussed in further detail in the Detailed Description section below. In this way, new bacterial targets can be identified that are inhibited by specific phage ORF products or derivatives, fragments, mimetics, or other molecules.
Other embodiments involve the identification and/or utilization of a target which is mutated at the site of phage 3A, 77 or 96 protein interaction but still functional in the cell by virtue of their host""s relatively unresponsive nature in the presence of expression of ORFs previously identified as inhibitory to the non-mutant or wild-type strain. Such mutants have the effect of protecting the host from an inhibition that would otherwise occur by, for example, competing for binding with the phage ORF product and indirectly allow identification of the precise responsible target. The identified target can then be used for, for example, follow-up studies and anti-microbial development. In certain embodiments, rescue and/or protection from inhibition occurs under conditions in which a bacterial target or mutant target is highly expressed. This is performed, for example, through coupling of the sequence with regulatory element promoters, as known in the art, which regulate expression at levels higher than wild-type at, for example, a level sufficiently higher than the inhibitor can be competitively bound to the highly expressed target such that the bacterium is detectably less inhibited.
Identification of the bacterial target can involve identification of a phage-specific site of action. This can involve a newly identified target, or a target where the phage site of action differs from the site of action of a previously known antibacterial agent or inhibitor. For example, phage T7 genes 0.7 and 2.0 target the host RNA polymerase, which is also the cellular target for the antibacterial agent, rifampin. To the extent that a phage product is found to act at a different site than previously described inhibitors, aspects of the present invention can utilize those new, phage-specific sites for identification and use of new agents. The site of action can be identified by techniques known to those skilled in the art, for example, by mutational analysis, binding competition analysis, and/or other appropriate techniques.
Once a bacterial host target or mutant target sequence has been identified, it too can be conveniently sequenced, sequence analyzed (e.g., by computer), and the underlying gene(s), and corresponding translated product(s) further characterized. Preferred embodiments include such analysis and identification. Preferably such a target has not previously been identified as an appropriate target for antibacterial action.
Also in preferred embodiments in which the bacterial target is a polypeptide or nucleic acid molecule, the identification of a bacterial target of a phage ORF product or fragment includes identification of a cellular and/or biochemical function of the bacterial target. As understood by those skilled in the art, this can, for example, include identification of function by identification of homologous polypeptides or nucleic acid molecules having known function, or identification of the presence of known motifs or sequences corresponding to known function. Such identifications can be readily performed using sequence comparison computer software, such as the BLAST programs and similar other programs and sequence and motif databases.
In embodiments involving expression of a phage ORF in a bacterial strain, in preferred embodiments that expression is inducible. By xe2x80x9cinduciblexe2x80x9d is meant that expression is absent or occurs at a low level until the occurrence of an appropriate environmental stimulus provides otherwise. For the present invention such induction is preferably controlled by an artificial environmental change, such as by contacting a bacterial strain population with an inducing compound (i.e., an inducer). However, induction could also occur, for example, in response to build-up of a compound produced by the bacteria in the bacterial culture, e.g., in the medium. As uncontrolled or constitutive expression of inhibitory ORFs can severely compromise bacteria to the point of eradication, such expression is therefore undesirable in many cases because it would prevent effective evaluation of the strain and inhibitor being studied. For example, such uncontrolled expression could prevent any growth of the strain following insertion of a recombinant ORF, thus preventing determination of effective transfection or transformation. A controlled or inducible expression is therefore advantageous and is generally provided through the provision of suitable regulatory elements, e.g., promoter/operator sequences that can be conveniently transcriptionally linked to a coding sequence to be evaluated. In most cases, the vector will also contain sequences suitable for efficient replication of the vector in the same or different host cells and/or sequences allowing selection of cells containing the vector, i.e., xe2x80x9cselectable markers.xe2x80x9d Further, preferred vectors include convenient primer sequences flanking the cloning region from which PCR and/or sequencing may be performed. In preferred embodiments where the purification of phage product is desired, preferably the bacterium or other cell type does not produce a target for the inhibitory product, or is otherwise resistant to the inhibitory product.
In preferred embodiments, the target of the phage ORF product or fragment is identified from a bacterial animal pathogen, preferably a mammalian pathogen, more preferably a human pathogen, and is preferably a gene or gene product of such a pathogen. Also in preferred embodiments, the target is a gene or gene product, where the sequence of the target is homologous to a gene or gene product from such a pathogen as identified above.
As used herein, the term xe2x80x9cmammalxe2x80x9d has its usual biological meaning, and particularly includes bovines, swine, dogs, cats, and humans.
Other aspects of the invention provide isolated, purified, or enriched specific phage nucleic acid and amino acid sequences, subsequences, and homologs thereof from or corresponding to ORFs 33, 41 and 79 from bacteriophage 3A, ORF 1 from bacteriophage 77 or ORFs 48, 78 and 100 from bacteriophage 96 (Staphylococcus aureus host bacterium). Such nucleotide sequences are at least 15 nucleotides in length, preferably at least 18, 21, 24, or 27 nucleotides in length, more preferably at least 30, 50, or 90 nucleotides in length. In certain embodiments, longer nucleic acids are preferred, for example those of at least 120, 150, 200, 300, 600, 900 or more nucleotides. Such sequences can, for example, be amplification oligonucleotides (e.g., PCR primers), oligonucleotide probes, sequences encoding a portion or all of a phage-encoded protein, or a fragment or all of a phage-encoded protein. In preferred embodiments, the nucleic acid sequence or amino acid sequence contains a sequence which has a lower length as specified above, and an upper-length limit which is no more than 50, 60, 70, 80, or 90% of the length of the full-length ORF or ORF product. The upper-length limit can also be expressed in terms of the number of base pairs of the ORF (coding region).
As it is recognized that alternate codons will encode the same amino acid for most amino acids due to the degeneracy of the genetic code, the sequences of this aspect includes nucleic acid sequences utilizing such alternate codon usage for one or more codons of a coding sequence. For example, all four nucleic acid sequences GCT, GCC, GCA, and GCG encode the amino acid, alanine. Therefore, if for an amino acid there exists an average of three codons, a polypeptide of 100 amino acids in length will, on average, be encoded by 3100, or 5xc3x971047, nucleic acid sequences. Thus, a nucleic acid sequence can be modified (e.g., a nucleic acid sequence from a phage as specified above) to form a second nucleic acid sequence encoding the same polypeptide as encoded by the first nucleic acid sequence using routine procedures and without undue experimentation. Thus, all possible nucleic acid sequences that encode the amino acid sequences encoded by the phage 3A ORFs 33, 41, and 79, the phage 77 ORF 1 and the phage 96 ORF 48, 78 and 100 as if all were written out in full, taking into account the codon usage, especially that preferred in the host bacterium.
The alternate codon descriptions are available in common textbooks, for example, Stryer, BIOCHEMISTRY 3rd ed., and Lehninger, BIOCHEMISTRY 3rd ed. Codon preference tables for various types of organisms are available in the literature. Because of the number of sequence variations involving alternate codon usage, for the sake of brevity, individual sequences are not separately listed herein. Instead the alternate sequences are described by reference to the natural sequence with replacement of one or more (up to all) of the degenerate codons with alternate codons from the alternate codon table (Table 2), preferably with selection according to preferred codon usage for the normal host organism or a host organism in which a sequence is intended to be expressed. Those skilled in the art also understand how to alter the alternate codons to be used for expression in organisms where certain codons code differently than shown in the xe2x80x9cuniversalxe2x80x9d codon table.
For amino acid sequences, sequences contain at least 5 peptide-linked amino acid residues, and preferably at least 6, 7, 10, 15, 20, 30, or 40, amino acids having identical amino acid sequence as the same number of contiguous amino acid residues in a phage 3A ORF 33, 41, or 79, or phage 77 ORF 1, or phage 96 ORF 48, 78, or 100 product. In some cases longer sequences may be preferred, for example, those of at least 50, 70, or 100 amino acids in length. In preferred embodiments, the sequence has bacteria-inhibiting function when expressed or otherwise present in a bacterial cell which is a host for the bacteriophage from which the sequence was derived.
By xe2x80x9cisolatedxe2x80x9d in reference to a nucleic acid is meant that a naturally occurring sequence has been removed from its normal cellular (e.g., chromosomal) environment or is synthesized in a non-natural environment (e.g., artificially synthesized). Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide chain present, but that it is essentially free (about 90-95% pure at least) of non-nucleotide material naturally associated with it, and thus is distinguished from isolated chromosomes.
The term xe2x80x9cenrichedxe2x80x9d means that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in cells from which the sequence was originally taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased.
The term xe2x80x9csignificantxe2x80x9d is used to indicate that the level of increase is useful to the person making such an increase and an increase relative to other nucleic acids of about at least 2-fold, more preferably at least 5- to 10-fold or even more. The term also does not imply that there is no DNA or RNA from other sources. The other source DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC19. This term distinguishes from naturally occurring events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
It is also advantageous for some purposes that a nucleotide sequence be in purified form. The term xe2x80x9cpurifiedxe2x80x9d in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation). Instead, it represents an indication that the sequence is relatively more pure than in the natural environment (compared to the natural level, this level should be at least 2-5 fold greater, e.g., in terms of mg/mL). Individual clones isolated from a genomic or cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones could be obtained directly from total DNA or from total RNA. cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 106-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. A genomic library can be used in the same way and yields the same approximate levels of purification.
The terms xe2x80x9cisolatedxe2x80x9d, xe2x80x9cenrichedxe2x80x9d, and xe2x80x9cpurifiedxe2x80x9d with respect to the nucleic acids, above, may similarly be used to denote the relative purity and abundance of polypeptides (multimers of amino acids joined one to another by xcex1-carboxyl:xcex1-amino group (peptide) bonds). These, too, may be stored in, grown in, screened in, and selected from libraries using biochemical techniques familiar in the art. Such polypeptides may be natural, synthetic or chimeric and may be extracted using any of a variety of methods, such as antibody immunoprecipitation, other xe2x80x9ctaggingxe2x80x9d techniques, conventional chromatography and/or electrophoretic methods. Some of the above utilize the corresponding nucleic acid sequence.
As indicated above, aspects and embodiments of the invention are not limited to entire genes and proteins. The invention also provides and utilizes fragments and portions thereof, preferably those which are xe2x80x9cactivexe2x80x9d in the inhibitory sense described above. Such peptides or oligopeptides and oligo or polynucleotides have preferred lengths as specified above for nucleic acid and amino acid sequences from phage; corresponding recombinant constructs can be made to express the encoded same. Also included are homologous sequences and fragments thereof.
The nucleotide and amino acid sequences identified herein are believed to be correct, however, certain sequences may contain a small percentage of errors, e.g., 1-5%. In the event that any of the sequences have errors, the corrected sequences can be readily provided by one skilled in the art using routine methods. For example, the nucleotide sequences can be confirmed or corrected by obtaining and culturing the relevant phage, and purifying phage genomic nucleic acids. A region or regions of interest can be amplified, e.g., by PCR from the appropriate genomic template, using primers based on the described sequence. The amplified regions can then be sequenced using any of the available methods (e.g., a dideoxy termination method, for example, using commercially available products). This can be done redundantly to provide the corrected sequence or to confirm that the described sequence is correct. Alternatively, a particular sequence or sequences can be identified and isolated as an insert or inserts in a phage genomic library and isolated, amplified, and sequenced by standard methods. Confirmation or correction of a nucleotide sequence for a phage gene provides an amino acid sequence of the encoded product by merely reading off the amino acid sequence according to the normal codon relationships and/or expressed in a standard expression system and the polypeptide product sequenced by standard techniques. The sequences described herein thus provide unique identification of the corresponding genes and other sequences, allowing those sequences to be used in the various aspects of the present invention. Confirmation of a phage ORF encoded amino acid sequence can also be confirmed by constructing a recombinant vector from which the ORF can be expressed in an appropriate host (e.g., E. coli), purified, and sequenced by conventional protein sequencing methods.
In other aspects the invention provides recombinant vectors and cells harboring phage 3A ORF 33, 41, or 79, or phage 77 ORF 1, or phage 96 ORF 48, 78, or 100 or portions thereof, or bacterial target sequences described herein, preferably where the phage or bacterial sequence is inserted in a recombinant vector. As understood by those skilled in the art, vectors may assume different forms, including, for example, plasmids, cosmids, and virus-based vectors. See, e.g., Maniatis, T. et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor University Press, Cold Spring, N.Y.; See also, Ausubel, F. M. et al. (eds.) (1994) Current Protocols in Molecular Biology. John Wiley and Sons, Secaucus, N.J.
In preferred embodiments, the vectors will be expression vectors, preferably shuttle vectors that permit cloning, replication, and expression within bacteria. An xe2x80x9cexpression vectorxe2x80x9d is one having regulatory nucleotide sequences containing transcriptional and translational regulatory information that controls expression of the nucleotide sequence in a host cell. Preferably the vector is constructed to allow amplification from vector sequences flanking an insert locus. In certain embodiments, the expression vectors may additionally or alternatively support expression, and/or replication in animal, plant and/or yeast cells due to the presence of suitable regulatory sequences, e.g., promoters, enhancers, 3xe2x80x2 stabilizing sequences, primer sequences, etc. In preferred embodiments, the promoters are inducible and specific for the system in which expression is desired, e.g., bacteria, animal, plant, or yeast. The vectors may optionally encode a xe2x80x9ctagxe2x80x9d sequence or sequences to facilitate protein purification or protein detection. Convenient restriction enzyme cloning sites and suitable selective marker(s) are also optionally included. Such selective markers can be, for example, antibiotic resistance markers or markers which supply an essential nutritive growth factor to an otherwise deficient mutant host, e.g., tryptophan, histidine, or leucine in the Yeast Two-Hybrid systems described below.
The term xe2x80x9crecombinant vectorxe2x80x9d relates to a single- or double-stranded circular nucleic acid molecule that can be transfected into cells and replicated within or independently of a cell genome. A circular double-stranded nucleic acid molecule can be cut and thereby linearized upon treatment with appropriate restriction enzymes. An assortment of nucleic acid vectors, restriction enzymes, and the knowledge of the nucleotide sequences cut by restriction enzymes are readily available to those skilled in the art. A nucleic acid molecule encoding a desired product can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together. Preferably the vector is an expression vector, e.g., a shuttle expression vector as described above.
By xe2x80x9crecombinant cellxe2x80x9d is meant a cell possessing introduced or engineered nucleic acid sequences, e.g., as described above. The sequence may be in the form of or part of a vector or may be integrated into the host cell genome. Preferably the cell is a bacterial cell.
In preferred embodiments, the inserted nucleic acid sequence corresponding to at least a portion of a phage 3A ORF 33, 41, and 79, phage 77 ORF 1 and phage 96 ORF48, 78, and 100 gene product has a length as specified for the isolated purified or enriched nucleic acid sequences in an aspect above.
In another aspect, the invention also provides methods for identifying and/or screening compounds xe2x80x9cactive onxe2x80x9d at least one bacterial target of a bacteriophage inhibitor protein or RNA. Preferred embodiments involve contacting bacterial target proteins with a test compound, and determining whether the compound binds to or reduces the level of activity of the bacterial target, e.g., a bacterial protein. Preferably this is done in vivo under approximately physiological conditions. The compounds that can be used may be large or small, synthetic or natural, organic or inorganic, proteinaceous or non-proteinaceous. In preferred embodiments, the compound is a peptidomimetic, as described herein, a bacteriophage inhibitor protein or fragment or derivative thereof, preferably an xe2x80x9cactiveportionxe2x80x9d, or a small molecule. In particular embodiments, the methods include the identification of bacterial targets as described above or otherwise described herein. Preferably the fragment of a bacteriophage inhibitor protein includes less than 80% of an intact bacteriophage inhibitor protein. Preferably, the at least one target includes a plurality of different targets of bacteriophage inhibitor proteins, preferably a plurality of different targets. The plurality of targets can be in or from a plurality of different bacteria, but preferably is from a single bacterial species.
In embodiments involving binding assays, preferably binding is to a fragment or portion of a bacterial target protein, where the fragment includes less than 90%, 80%, 70%, 60%, 50%, 40%, or 30% of an intact bacterial target protein. Preferably, the at least one bacterial target includes a plurality of different targets of bacteriophage inhibitor proteins, preferably a plurality of different targets. The plurality of targets can be in or from a plurality of different bacteria, but preferably is from a single bacterial species.
A xe2x80x9cmethod of screeningxe2x80x9d refers to a method for evaluating a relevant activity or property of a large plurality of compounds, rather than just one or a few compounds. For example, a method of screening can be used to conveniently test at least 100, more preferably at least 1000, still more preferably at least 10,000, and most preferably at least 100,000 different compounds, or even more.
In the context of this invention, the term xe2x80x9csmall moleculexe2x80x9d refers to compounds having molecular mass of less than 3000 Daltons, preferably less than 2000 or 1500, still more preferably less than 1000, and most preferably less than 600 Daltons. Preferably but not necessarily, a small molecule is not an oligopeptide.
In a related aspect or in preferred embodiments, the invention provides a method of screening for potential antibacterial agents by determining whether any of a plurality of compounds, preferably a plurality of small molecules, is active on at least one target of a bacteriophage inhibitor protein or RNA. Preferred embodiments include those described for the above aspect, including embodiments which involve determining whether one or more test compounds bind to or reduce the level of activity of a bacterial target, and embodiments which utilize a plurality of different targets as described above.
The identification of bacteria-inhibiting phage ORFs and their encoded products also provides a method for identifying an active portion of such an encoded product. This also provides a method for identifying a potential antibacterial agent by identifying such an active portion of a phage ORF or ORF product. In preferred embodiments, the identification of an active portion involves one or more of mutational analysis, deletion analysis, or analysis of fragments of such products. The method can also include determination of a 3-dimensional structure of an active portion, such as by analysis of crystal diffraction patterns. In further embodiments, the method involves constructing or synthesizing a peptidomimetic compound, where the structure of the peptidomimetic compound corresponds to the structure of the active portion.
In this context, xe2x80x9ccorrespondsxe2x80x9d means that the peptidomimetic compound structure has sufficient similarities to the structure of the active portion that the peptidomimetic will interact with the same molecule as the phage protein and preferably will elicit at least one cellular response in common which relates to the inhibition of the cell by the phage protein.
The methods for identifying or screening for compounds or agents active on a bacterial target of a phage-encoded inhibitor can also involve identification of a phage-specific site of action on the target.
An xe2x80x9cactive portionxe2x80x9d as used herein denotes an epitope, a catalytic or regulatory domain, or a fragment of a bacteriophage inhibitor protein that is responsible for, or a significant factor in, bacterial target inhibition. The active portion preferably may be removed from its contiguous sequences and, in isolation, still effect inhibition.
By xe2x80x9cmimeticxe2x80x9d is meant a compound structurally and functionally related to a reference compound that can be natural, synthetic, or chimeric. In terms of the present invention, a xe2x80x9cpeptidomimetic,xe2x80x9d for example, is a compound that mimics the activity-related aspects of the 3-dimensional structure of a peptide or polypeptide in a non-peptide compound, for example mimics the structure of a peptide or active portion of a phage- or bacterial ORF-encoded polypeptide.
A related aspect provides a method for inhibiting a bacterial cell by contacting the bacterial cell with a compound active on a bacterial target of a bacteriophage inhibitor protein or RNA encoded by bacteriophage 3A ORF 33, 41, or 79, bacteriophage 77 ORF 1, or bacteriophage 96 ORF 48, 78, or 100, where the target was uncharacterized. In preferred embodiments, the compound is such a protein, or a fragment or derivative thereof; a structural mimetic, e.g., a peptidomimetic, of such a protein or fragment; a small molecule; the contacting is performed in vitro, the contacting is performed in vivo in an infected or at risk organism, e.g., an animal such as a mammal or bird, for example, a human, or other mammal described herein, or in a plant.
In the context of this invention, the term xe2x80x9cbacteriophage inhibitor proteinxe2x80x9d refers to a protein encoded by a bacteriophage nucleic acid sequence which inhibits bacterial function in a host bacterium. Thus, it is a bacteria-inhibiting phage product.
In the context of this invention, the phrase xe2x80x9ccontacting the bacterial cell with a compound active on a bacterial target of a bacteriophage inhibitor proteinxe2x80x9d or equivalent phrases refer to contacting with an isolated, purified, or enriched compound or a composition including such a compound, but specifically does not rely on contacting the bacterial cell with an intact naturally occurring phage which encodes the compound. Preferably no intact phage are involved in the contacting.
Related aspects provide methods for prophylactic or therapeutic treatment of a bacterial infection by administering to an infected, challenged or at risk organism a therapeutically or prophylactically effective amount of a compound active on a target of a bacteriophage 3A ORF 33, 41, or 79, bacteriophage 77 ORF 1, or bacteriophage 96 ORF 48, 78, or 100 product, e.g., as described for the previous aspect. Preferably the bacterium involved in the infection or risk of infection produces the identified target of the bacteriophage inhibitor protein or alternatively produces a homologous target compound. In preferred embodiments, the host organism is a plant or animal, preferably a mammal or bird, and more preferably, a human or other mammal described herein. Preferred embodiments include, without limitation, those as described for the preceding aspect.
Compounds useful for the methods of inhibiting, methods of treating, and pharmaceutical compositions can include novel compounds, but can also include compounds which had previously been identified for a purpose other than inhibition of bacteria. Such compounds can be utilized as described and can be included in pharmaceutical compositions.
By xe2x80x9ctreatmentxe2x80x9d or xe2x80x9ctreatingxe2x80x9d is meant administering a compound or pharmaceutical composition for prophylactic and/or therapeutic purposes. The term xe2x80x9cprophylactic treatmentxe2x80x9d refers to treating a patient or animal that is not yet infected but is susceptible to or otherwise at risk of a bacterial infection. The term xe2x80x9ctherapeutic treatmentxe2x80x9d refers to administering treatment to a patient already suffering from infection.
The term xe2x80x9cbacterial infectionxe2x80x9d refers to the invasion of the host organism, animal or plant, by pathogenic bacteria. This includes the excessive growth of bacteria which are normally present in or on the body of the organism, but more generally, a bacterial infection can be any situation in which the presence of a bacterial population(s) is damaging to a host organism. Thus, for example, an organism suffers from a bacterial infection when excessive numbers of a bacterial population are present in or on the organism""s body, or when the effects of the presence of a bacterial population(s) is damaging to the cells, tissue, or organs of the organism.
The terms xe2x80x9cadministerxe2x80x9d, xe2x80x9cadministeringxe2x80x9d, and xe2x80x9cadministrationxe2x80x9d refer to a method of giving a dosage of a compound or composition, e.g., an antibacterial pharmaceutical composition, to an organism. Where the organism is a mammal, the method is, e.g., topical, oral, intravenous, transdermal, intraperitoneal, intramuscular, or intrathecal. The preferred method of administration can vary depending on various factors, e.g., the components of the pharmaceutical composition, the site of the potential or actual bacterial infection, the bacterium involved, and the infection severity.
The term xe2x80x9cmammalxe2x80x9d has its usual biological meaning, referring to any organism of the Class Mammalia of higher vertebrates that nourish their young with milk secreted by mammary glands, e.g., mouse, rat, and, in particular, human, bovine, sheep, swine, dog, and cat.
In the context of treating a bacterial infection a xe2x80x9ctherapeutically effective amountxe2x80x9d or xe2x80x9cpharmaceutically effective amountxe2x80x9d indicates an amount of an antibacterial agent, e.g., as disclosed for this invention, which has a therapeutic effect. This generally refers to the inhibition, to some extent, of the normal cellular functioning of bacterial cells that renders or contributes to bacterial infection.
The dose of antibacterial agent that is useful as a treatment is a xe2x80x9ctherapeutically effective amount.xe2x80x9d Thus, as used herein, a therapeutically effective amount means an amount of an antibacterial agent that produces the desired therapeutic effect as judged by clinical trial results and/or animal models. This amount can be routinely determined by one skilled in the art and will vary depending on several factors, such as the particular bacterial strain involved and the particular antibacterial agent used.
In connection with claims to methods of inhibiting bacteria and therapeutic or prophylactic treatments, xe2x80x9ca compound active on a target of a bacteriophage inhibitor proteinxe2x80x9d or terms of equivalent meaning differ from administration of or contact with an intact phage naturally encoding the full-length inhibitor compound. While an intact phage may conceivably be incorporated in the present methods, the method at least includes the use of an active compound as specified different from a full length inhibitor protein naturally encoded by a bacteriophage and/or a delivery or contacting method different from administration of or contact with an intact phage naturally encoding the full-length protein. Similarly, pharmaceutical compositions described herein at least include an active compound or composition different from a phage naturally coding the full-length inhibitor protein, or such a full-length protein is provided in the composition in a form different from being encoded by an intact phage. Preferably the methods and compositions do not include an intact phage.
In accordance with the above aspects, the invention also provides antibacterial agents and compounds active on a bacterial target of bacteriophage 3A ORF 33, 41, or 79, bacteriophage 77 ORF 1, or bacteriophage 96 ORF 48, 78, or 100, where the target was uncharacterized as indicated above. As previously indicated, such active compounds include both novel compounds and compounds which had previously been identified for a purpose other than inhibition of bacteria. Such previously identified biologically active compounds can be used in embodiments of the above methods of inhibiting and treating. In preferred embodiments, the targets, bacteriophage, and active compound are as described herein for methods of inhibiting and methods of treating. Preferably the agent or compound is formulated in a pharmaceutical composition which includes a pharmaceutically acceptable carrier, excipient, or diluent. In addition, the invention provides agents, compounds, and pharmaceutical compositions where an active compound is active on an uncharacterized phage-specific site on the target.
In preferred embodiments, the target is as described for embodiments of aspects above.
Likewise, the invention provides a method of making an antibacterial agent. The method involves identifying a target of a bacteriophage 3A ORF 33, 41, or 79, bacteriophage 77 ORF 1, or bacteriophage 96 ORF 48, 78, or 100 product, screening a plurality of compounds to identify a compound active on the target, and synthesizing the compound in an amount sufficient to provide a therapeutic effect when administered to an organism infected by a bacterium naturally producing the target.
In preferred embodiments, the identification of the target and identification of active compounds include steps or methods and/or components as described above (or otherwise herein) for such identification. Likewise, the active compound can be as described above, including fragments and derivatives of phage inhibitor proteins, peptidomimetics, and small molecules. As recognized by those skilled in the art, peptides can be synthesized by expression systems and purified, or can be synthesized artificially by methods well known in the art.
In the context of nucleic acid or amino acid sequences of this invention, the term xe2x80x9ccorrespondingxe2x80x9d and xe2x80x9ccorrespondxe2x80x9d indicates that the sequence is at least 95% identical, preferably at least 97% identical, and more preferably at least 99% identical to a sequence from the specified phage genome or bacterial genome, a ribonucleotide equivalent, a degenerate equivalent (utilizing one or more degenerate codons), or a homologous sequence, where the homolog provides functionally equivalent biological function.
In embodiments where the bacterial target of a bacteriophage inhibitor ORF product, e.g., an inhibitory protein or polypeptide, the target is preferably encoded by a S. aureus nucleic acid coding sequence from a host bacterium for bacteriophages 3A, 77, or 96. Target sequences are described herein by reference to sequence source sites. The sequence encoding the target preferably corresponds to a S. aureus nucleic acid sequence available from numerous sources including S. aureus sequences deposited in GenBank, S. aureus sequences found in European Patent Application No. 97100110.7 to Human Genome Sciences, Inc. filed Jan. 7, 1997, S. aureus sequences available from TIGR at http://www.tigr.org/tdb/mdb/mdb.html, and S. aureus sequences available from the Oklahoma University S. aureus sequencing project at the following URL:
http://www.genome.ou.edu/staph_new.html.
The amino acid sequence of a polypeptide target is readily provided by translating the corresponding coding region. For the sake of brevity, the sequences are not reproduced herein. Also, in preferred embodiments, a target sequence corresponds to a S. aureus coding sequences corresponding to a sequence listed in Table 7. The listings in Table 7 describe S. aureus sequences currently deposited in GenBank. Again, for the sake of brevity, the sequences are described by reference to the GenBank entries instead of being written out in full herein. In cases where an entry for a coding region is not complete, the complete sequence can be readily obtained by routine methods, by isolating a clone in a phages 3A, 77, and 96 host S. aureus genomic library, and sequencing the clone insert to provide the relevant coding region. The boundaries of the coding region can be identified by conventional sequence analysis and/or by expression in a bacterium in which the endogenous copy of the coding region has been inactivated and using subcloning to identify the functional start and stop codons for the coding region.
In an additional aspect, the present invention provides a nucleic acid segment which encodes a protein and corresponds to a segment of the nucleic acid sequence of an ORF (open reading frame) from Staphylococcus aureus bacteriophages 3A, 77 or 96 as provided in Table 1. Preferably, the protein is a functional protein. One of ordinary skill in the art would recognize that bacteriophage possess genes which encode proteins which may be either beneficial or detrimental to a bacterial cell. Such proteins act to replicate DNA, translate RNA, manipulate DNA or RNA, and enable the phage to integrate into the bacterial genome. Proteins from bacteriophage can function as, for example, a polymerase, kinase, phosphatase, helicase, nuclease, topoisomerase, endonuclease, reverse transcriptase, endoribonuclease, dehydrogenase, gyrase, integrase, carboxypeptidase, proteinase, amidase, transcriptional regulators and the like, and/or the protein may be a functional protein such as a chaperon, capsid protein, head and tail proteins, a DNA or RNA binding protein, or a membrane protein, all of which are provided as non-limiting examples. Proteins with functions such as these are useful as tools for the scientific community.
Thus, the present invention provides a group of novel proteins from bacteriophage which can be used as tools for biotechnical applications such as, for example, DNA and/or RNA sequencing, polymerase chain reaction and/or reverse transcriptase PCR, cloning experiments, cleavage of DNA and/or RNA, reporter assays and the like. Preferably, the protein is encoded by an open reading frame in the nucleic acid sequences of bacteriophages 3A, 77 or 96. Within the scope of the present invention are fragments of proteins and/or truncated portions of proteins which have been either engineered through automated protein synthesis, or prepared from nucleic acid segments which correspond to segments of the nucleic acid sequences of bacteriophages 3A, 77 or 96, and which are then inserted into cells via plasmid vectors which can be induced to express the protein. It is understood by one of skill in the art that mutational analysis of proteins has been known to help provide proteins which are more stable and which have higher and/or more specific activities. Such mutations are also within the scope of the present invention, hence, the present invention provides a mutated protein and/or the mutated nucleic acid segment from bacteriophages 3A, 77 or 96 which encodes the protein.
In another aspect, the invention provides antibodies which bind proteins encoded by a nucleic acid segment which corresponds to the nucleic acid sequence of an ORF (open reading frame) from Staphylococcus aureus bacteriophages 3A, 77 or 96 as provided in Table 1. Bacteriophages are bacterial viruses which contain nucleic acid sequences which encode proteins that can correspond to proteins of other bacteriophages and other viruses. Antibodies targeted for proteins encoded by nucleic acid segments of phages 3A, 77 or 96 can serve to bind targets encoded by nucleic acid segments from other viruses which correspond to the sequences provided in Table 1. Furthermore, antibodies to proteins encoded by nucleic acid segments of phages 3A, 77 or 96 can also bind to proteins from other viruses that share similar functions but may not share corresponding sequences. It is understood in the art that proteins with similar activities/functions from a variety of sources generally share motifs, regions, or domains which correspond. Thus, antibodies to motifs, regions, or domains of functional proteins from phages 3A, 77 or 96 should be useful in detecting corresponding proteins in other bacteriophages and viruses. Such antibodies can also be used to detect the presence of a virus sharing a similar protein. Preferably the virus to be detected is pathogenic to a mammal, such as a dog, cat, bovine, sheep, swine, or a human.
As used in the claims to describe the various inventive aspects and embodiments, xe2x80x9ccomprisingxe2x80x9d means including, but not limited to, whatever follows the word xe2x80x9ccomprisingxe2x80x9d. Thus, use of the term xe2x80x9ccomprisingxe2x80x9d indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By xe2x80x9cconsisting ofxe2x80x9d is meant including, and limited to, whatever follows the phrase xe2x80x9cconsisting ofxe2x80x9d. Thus, the phrase xe2x80x9cconsisting ofxe2x80x9d indicates that the listed elements are required or mandatory and that no other elements may be present. By xe2x80x9cconsisting essentially ofxe2x80x9d is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase xe2x80x9cconsisting essentially ofxe2x80x9d indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
Additional features and embodiments of the present invention will be apparent from the following Detailed Description and from the claims, all within the scope of the present invention.